It is known in the art to produce fusion proteins for a number of purposes. In some cases, the two protein units in the single polypeptide have two essentially independent activities. The most common example of this application is the fusion of marking proteins, such as GFP, to intracellular factors as a means of observing their localization and expression (see, for example, A. W. Kerrebrock et al., Cell, 83:247–56, 1995; H. G. Wang et al., Cell, 87:629–638, 1996). Creation of fusion proteins has also been used to prolong the half-life of a protein (see, for example, R. A. Hallewell, et al., J. Biol. Chem., 264:5260–5268, 1989; T. P. Yao et al., Cell, 77:6372, 1992) as well as other uses (see, for example, T. Sano et al., Proc Natl Acad Sci U.S.A., 89:1534–1538, 1992).
A more complicated application of protein fusion is the production of fusion proteins wherein the two protein units cooperate to achieve a biological function. In functional dimers, both proteins must fold and interact with each other appropriately. V. A. Garcia-Campayo et al. (Nature Biotech, 15:663–667, (1997)) have utilized a peptide linker to fuse gene subunits together into a single biologically active peptide. Neuhold and Wold, (Cell, 74:1033–1042, (1993)) have reported the fusion of two proteins into a single biologically active protein that binds DNA targets, wherein the protein units interact with each other to the exclusion of competing heterodimer partners. However, fusion of proteins with multiple functions has been more difficult to produce, for example, steroid/thyroid hormone nuclear receptors are complex, multifunctional proteins with, minimally, four interconnected yet separable functions: ligand binding, dimerization, DNA binding, and transactivation.
Steroid/thyroid hormone nuclear receptors are used in the field of genetic engineering as a tool for studying control of gene expression and to manipulate and control development and other physiological processes. For example, applications for regulated gene expression in mammalian systems include inducible gene targeting, overexpression of toxic and teratogenic genes, anti-sense RNA expression, and gene therapy (see, for example, R. Jaenisch, Science 240:1468–1474, 1988). For cultured cells, glucocorticoids and other steroids have been used to induce the expression of a desired gene.
As another means for controlling gene expression in mammalian systems, an inducible tetracycline regulated system has been devised and utilized in transgenic mice, whereby gene activity is induced in the absence of tetracycline and repressed in its presence (see, e.g, Gossen et al. PNAS 89:5547–5551, 1992; Gossen et al., TIBS 18:471–475, 1993; Furth et al., PNAS 91:9302–9306, 1994; and Shockett et al., PNAS 92:6522–6526, 1995). However, disadvantages of the inducible tetracycline system include the requirement for continuous administration of tetracycline to repress expression and the slow clearance of antibiotic from bone, a side-effect that interferes with regulation of gene expression. While this system has been improved by the recent identification of a mutant tetracycline repressor that acts conversely as an inducible activator, the pharmacokinetics of tetracycline may hinder its use during development when a precise and efficient “on-off” switch is essential (see, e.g., Gossen et al., Science 268:1766–1769, 1995).
Certain insect steroid/thyroid hormone nuclear receptors have also been studied. The Drosophila melanogaster ecdysone receptor (EcR) (M. R. Koelle et al., Cell 67:59–77, 1995) is unlike the estrogen, androgen, and other homodimeric vertebrate steroid hormone nuclear receptors because it requires a heterologous dimer partner for functional transactivation. The obligate dimer partner, the product of the ultraspiracle (Usp) gene (V. C. Henrich et al., Nuc. Acids Res. 18: 4143–4148, 1990; T. P. Yao et al., supra, 1992; T. P. Yao et al., Nature 366:476–479, 1993), is an insect homolog of the mammalian retinoid X receptor (RXR) proteins found in vertebrates and other mammalian species. RXRs have been characterized as regulatory dimer partners of many mammalian class II steroid/thyroid hormone nuclear receptors, such as the thyroid hormone receptors, the retinoic acid receptors, and the vitamin D receptor (reviewed in Mangelsdorf and Evans, Cell 83:841–850, 1995; D. J. Mangelsdorf et al., Cell 83: 835–839, 1995). RXR is also a dimer partner of EcR.
Usp and RXR share a significant degree of sequence homology and some functional similarities; however, in formation of heterodimers with EcR, RXR interacts differently than Usp. One primary difference is that formation of EcR+RXR heterodimers is more highly stimulated by the steroid ligand ecdysteroid muristerone A (murA) than by 20-hydroxyecdysone (20-Ec), while formation of EcR+Usp heterodimers is potently stimulated by 20-hydroxyecdysone (K. S. Christopherson et al., Proc Natl Acad Sci USA 89:6314–6318, 1982; H. E. Thomas et al., Nature 362:471–475, 1993). A second difference is in the way that ligand promotes efficient formation of EcR+Usp and EcR+RXR heterodimer complexes and concomitant binding to ecdysone response elements (EcREs). MurA stimulates EcR+Usp binding of EcREs approximately 3 to 7-fold over levels without ligand, but EcR+RXR complexes are completely dependent on ligand for heterodimerization. Further EcR+RXR complexes bind to EcREs at only 10–40% the level of EcR+Usp complexes (Christopherson et al., supra 1982; Thomas et al., supra 1993; Yao et al., supra, 1992 & 1993). This suggests that the affinity of EcR for its natural dimer partner, Usp, is significantly greater than its affinity for RXR.
EcR has been studied for use in transgene regulation; however, its use for this purpose is complicated by the requirement for superphysiological levels of RXR protein to be coexpressed (No et al., supra 1997), presumably because of the comparatively low affinity of EcR for RXR as a dimer partner. Of the mammalian cell types heretofore examined, only the 293 cell line appears capable of supporting high level transactivation of EcR without added RXR (Christopherson et al., supra, 1982). The requirement for co-expression of RXR in most mammalian systems raises concerns that RXR will heterodimerize with endogenous mammalian class II steroid/thyroid hormone nuclear receptors, causing altered differentiation, growth, or fitness of transduced cells.
A number of ecdysone receptors are known in the art as being a gene sequence responsive to an applied exogenous chemical inducer enabling external control of expression of the gene controlled by the receptor (See, for example, PCT/GB96/01195).
Accordingly, there is a need in the art for improved systems to precisely modulate the expression of exogenous genes in mammalian subjects. For example, a non-mammalian-based transcription regulating system would be extremely desirable for general application to transgene regulation in in vitro, ex vivo, and in vivo applications. In addition, there is a need in the art for new and better methods of using steroid/thyroid hormone nuclear receptors that require a dimer partner for functional transactivation of transgene expression for use in somatic gene therapy and for laboratory models thereof.